R-loops promote antisense transcription across the mammalian genome

We report the correlation we found between R-loops and antisense trasncription initiation. Our in vitro data suggests that RNA pol II can initiate transcription off the displaced single stranded DNA from within the R-loop. Therefore we generated Chromatin associated RNA-seq with and without over-expressing RNaseH1 and RDIP-seq (the directional RNA-seq of the RNA moeity from within the S9.6 pull down R-loop) as well as RR-ChIP-seq (the directional RNA-seq of the RNA moeity from within the catalytically dead RNaseH1-GFP GFP pull down R-loop) and confirm the correlation in vivo. We also aligned our findings with ChrCAP-seq (which is is a library enriched for RNA polymerase II capped RNA using chromatin associated RNA as the initial source) and found that capped antisense RNA were sensitive to RNase H1 overexpression. Examination of chromatin associated RNA of HeLa cells and capped RNA with and without over-expressing RNAseH1. Directional R-loop occupancy was conducted by 1) sequencing the RNA moeity of the R-loop fragments which were immunoprecipitated by mouse monoclonal S9.6 antibody and by 2) sequencing the RNA moeity of the RNA:DNA hybrids caught in a catalytically dead RNaseH1-GFP.